Role of NF-κB in hematopoietic niche function of osteoblasts after radiation injury

Abstract

Hematopoietic tissue is very sensitive to ionizing radiation (IR). In adult mammalian bone marrow, hematopoietic stem and progenitor cells (HSPC) reside next to the endosteal bone surface, which is lined primarily by osteoblastic cells. In the present study, we proposed to investigate the mechanisms by which osteoblasts in the hematopoietic niche regulate survival, proliferation, and differentiation of HSPC after radiation injury. Human primary CD34+ HSPC were cultured with human fetal osteoblast (hFOB) cell line cells or conditioned medium (CM) from hFOB cells with or without irradiation. Survival, apoptosis, and cell cycle were analyzed using clonogenic and flow cytometric assays. Cytokine and chemokine expression were measured by cytokine array and enzyme-linked immunosorbent assay. Their regulatory activities were assessed by quantitative real-time polymerase chain reaction, small interfering (si)RNA transfection, immunoblotting, and transbinding assays. Survival of gamma-irradiated CD34+ HSPC was significantly enhanced by coculture with hFOB cells or by CM from hFOB cells. There were six factors in hFOB cell lysates and five factors released into hFOB CM enhanced by IR. IR induced phosphorylation of p53, c-Jun, and p38 and downstream p21 expression, as well as cell cycle arrest and apoptosis in hFOB cells. However, IR also induced phosphorylation of nuclear factor (NF)-kappaBp65 (ser536) and NF-kappaB activation in hFOB cells. Inhibition of NF-kappaB expression with siRNA upregulated p21, inhibited release of cytokines and chemokines, and induced hFOB and CD34+ cell apoptosis. NF-kappaB is a radiation-induced prosurvival factor in human osteoblastic cells. NF-kappaB gene knockdown abrogated the hematopoietic niche function of hFOB cells in supporting survival of CD34+ cells after IR.